1. Field of the Invention
The present invention generally relates to antibody and more particularly, relates to variable regions of heavy and light chains of an antibody specific to a surface antigen in sporozoite of Eimeria spp. and scFV (single chain variable fragment) prepared using the variable regions.
2. Description of the Related Art
Avian coccidiosis, caused by intestinal parasites belonging to genus Eimeria, is an obligate protozoan disease of chickens, resulting in a significant economic loss in the poultry industry. Despite increasing interest in developing protection strategies, the use of whole parasites or chemotherapy has major drawbacks. For example, due to the complexity of the parasite life-cycle and the existence of multiple species infecting chickens, immunity developed by using whole parasites, in general, is species-specific and cross-species protection has not been observed (Reynaud, C. A. et al., Eur. J. Immunol. 21:2661(1991)). The application of anti-coccidia drugs is also hindered by high costs and development of drug resistance. Therefore, research has been focused on the development of immunological controls, which is dependent on the identification and characterization of target antigens to induce protective immune responses by the host immune system.
Current efforts to develop an immunological control against coccidiosis involve identification of immunogenic epitope of Eimeria parasites to elicit cell mediated immunity (Lillehoj, H. S. et al., Avian Dis., 44:408-425(2000)). In general, two immunological strategies have been envisioned. The first uses recombinant subunit vaccines derived from parasite proteins used to bind to host cell receptors since avian coccidian parasites are known to invade cells of intestinal surface epithelium (Al-Attar, M. A. et al., J. Parasitol., 73:494-502(1987); and Lawn, A. M. et al., J. Parasitol., 68:1117-1123 (1982)). The second approach involves passive immunization with antibodies that actively block the interaction of parasites with host cells (Sasaki, K. et al., J. Parasitol., 82:82-87(1996)).
Many coccidial antigens have been identified with mouse antibodies (Speer, C. A. et al., J. Protozol., 30:548-554(1983)), and their cDNAs have been cloned for the development of a subunit vaccine (Castle, M. D. et al., J. Parasitol., 77:384-390(1991); and Ko, C. et al., Mol. Bio. Parasitol., 73:790-792 (1993)). However, the efficacy of these antibodies is debatable (Trout, J. et al., J. Parasitol., 73:790-792(1993)), because of differences in the target antigens recognized by immune sera from chickens and mice (Jenkins, M. C. et al., Mol. Bio. Parasitol., 25:155-164(1987)).
Therefore, in this regard, chicken antibodies may be more advantageous for the identification of target antigens to cause avian coccidiosis.
Recently, the present inventors have been developed four chicken monoclonal antibodies (Mabs: 2-1, 5D11, 8C3 and 13C8) which recognize Eimeria antigens (Lillehoj, H. S. et al., Eimeria. Poul. Sci., 73:1685-1693(1994) and Lillehoj, H. S. et al., J. Parasitol., 82:82-87(1996)), and characterized their biochemical properties. The immunologic nature of antigens recognized by these antibodies is under the investigation. Recently, the present inventors found that the developed chicken Mabs (monoclonal antibodies) recognize the surface antigens localized in the apical complex of Eimeria acervulina. This promising result suggests the possible application of anti-Eimeria Mabs for passive immunization. However, chicken hybridomas have some drawbacks such as production of a low amount of antibody and of non-specific IgM, and the loss of ability to produce antibodies (Nishinaka, S. et al., J. Immunol. Methods., 139:217-222(1991); and Nishinaka, S. et al., J. Vet. Med. Sci., 58:1053-1056(1996)).
U.S. Pat. No. 4,710,377 discloses monoclonal antibodies against sporozoites of he Eimeria spp. obtained by use of hybridoma technology, and U.S. Pat. No. discloses novel recombinant antigenic proteins of avian coccidiosis, and fragments thereof containing antigenic determinants.
Moreover, U.S. Pat. No. 5,635,181 discloses anti-coccidial vaccine containing a recombinant peptide with novel epitopes and U.S. Pat. No. 4,301,148 discloses a method for preventing fowl coccidiosis comprising inoculating newly hatched fowl with sporozoites of Eimeria.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.
In one aspect of this invention, there is provided a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 38.
In another aspect of this invention, there is provided a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 40.
In still another aspect of this invention, there is provided a DNA molecule encoding a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 38.
In further aspect of this invention, there is provided a DNA molecule encoding a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 40.
In still further aspect of this invention, there is provided a recombinant scFv (single chain variable fragment) antibody specific to a surface antigen in sporozoite of Eimeria spp., which comprises: (a) a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 38; and (b) a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 40.
In another aspect of this invention, there is provided a DNA molecule encoding scFv antibody specific to a surface antigen in sporozoite of Eimeria spp., which comprises: (a) a DNA molecule encoding a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 38; and (b) a DNA molecule encoding a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 40.
In still another aspect of this invention, there is provided a method for preparing a recombinant scFv antibody specific to a surface antigen in sporozoite of Eimeria spp., which comprises: (a) cloning an scFv gene construct comprising (i) a DNA molecule encoding a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 38; and (ii) a DNA molecule encoding a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 40 into an expression vector; (b) transforming host cells with the expression vector of (a); and (c) expressing and isolating the recombinant scFv antibody in host cells.
In further aspect of this invention, there is provided an expression vector for expressing a recombinant scFv antibody specific to a surface antigen in sporozoite of Eimeria spp., which comprises: (a) an scFv gene construct comprising (i) a DNA molecule encoding a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 38; and (ii) a DNA molecule encoding a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp., wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 40 into an expression vector; and (b) a promoter controlling an expression of scFv gene construct.
Accordingly, it is an object of this invention to provide a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is another object of this invention to provide a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is still another object of this invention to provide a DNA molecule encoding a heavy chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is further object of this invention to provide a DNA molecule encoding a light chain variable region of an antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is still further object of this invention to provide a recombinant scFv antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is another object of this invention to provide a DNA molecule encoding scFv antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is still another object of this invention to provide a method for preparing a recombinant scFv antibody specific to a surface antigen in sporozoite of Eimeria spp.
It is further object of this invention to provide an expression vector for expressing a recombinant scFv antibody specific to a surface antigen in sporozoite of Eimeria spp.
Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.